Multivariate resolution methods make up a set of mathematical tools that may be applied to the analysis and interpretation of spectroscopic data recorded when monitoring a physical or chemical process with multichannel detectors. The goal of resolution methods is the recovery of chemical and/or physical information from the experimental data. Such data include, for example, the number of intermediates present in a reaction, the rate or equilibrium constants, and the spectra for each one of those intermediates. Multivariate resolution methods have been shown to be useful for the study of biophysical and biochemical processes such as folding/unfolding of proteins or nucleic acids. The present article reviews the most frequently used resolution methods, the limitations on their use, and their latest applications in protein and nucleic acid research.